Numerous insects are serious pests of common agricultural crops. One method of controlling insects has been to apply insecticidal organic, semiorganic or organometallic chemicals to crops. This method has numerous, art-recognized environmental and public health problems. A more recent method of control of insect pests has been the use of biological control organisms which are typically natural predators of the troublesome insects. These include other insects such as trachonid wasps, fungi such as Beauveria bassiana, and bacteria such as Bacillus thuringiensis cv., commonly referred to as "Bt". However, it is difficult to apply biological control organisms to large areas, and even more difficult to cause those living organisms to remain in the treated area for an extended period. Still more recently, techniques in recombinant DNA have provided the opportunity to insert into plant cells cloned genes which express insecticidal toxins derived from biological control organisms such as Bt. This technology has given rise to concerns about eventual insect resistance to well-known, naturally occurring insect toxins, particularly in the face of heavy selection pressure, which may occur in some areas. Thus, a continuing need exists to identify naturally occurring insecticidal toxins which can be formed by plant cells directly by expression of structural genes not normally present in the plant.
Southern Corn Rootworm (Diabrotica undecimpunctata howardi Barber) is a particularly difficult pest to control or eradicate. It attacks the plant below the soil line, where insecticides are difficult or impossible to apply effectively. In addition, it is resistant to a number of otherwise effective chemical and biological control agents, including Bt toxins and some lectins.
The monoterpene, limonene, 1-methyl-4 (1-methylethenyl) cyclohexene; p-mentha-1,8-diene (Entry No. 5371, Merck Index 11th Ed.), occurs naturally in various ethereal oils, particularly oils of lemon, orange, caraway, dill and bergamot. It is a valuable industrial chemical. Some limonene is prepared by extraction from plants of the mint family, a large quantity is obtained from citrus oils, which are typically 80-90% limonene, and some is obtained from pine oil. It is also synthesized chemically and finds use as a solvent and cleaning agent (in the manufacture of synthetic pine oil), as an expectorant, as a wetting and dispersing agent, as a monomer in the manufacture of various polymeric resins, as a flavorant and a precursor in the synthesis of the flavorant carvone, and as a polymerization inhibitor in storage of the tetrafluoreoethylene monomer used in the manufacture of polytetrafluoroethylene (PTFE).
Geranyl diphosphate (GPP) synthase catalyzes the first committed step of monoterpene biosynthesis by the condensation of dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) to form GPP, the immediate acyclic precursor of monoterpenes. GPP is converted to (-)-4S-limonene by the catalytic action of (-)-4S-limonene synthase (cyclase). (-)-4S-limonene is converted to (-)-trans-carveol by the action of (-)-trans-carveol synthase, also referred to as limonene-6-hydroxylase. -(-)trans-carveol is converted to carvone by the action of -(-)trans-carveol dehydrogenase, also referred to as carvone synthase. GPP is also converted to the monoterpene S-linalool by the action of S-linalool synthase.
Thus, GPP is the precursor of (-)-4S-limonene and its downstream metabolites (-)-trans-carveol and carvone; as well as the precursor of S-linalool. See FIG. 1; Wise et al. (1997) In "Comprehensive Natural Products Chemistry: Isoprenoids, Vol. 2" (Cane, D. E., ed.), Elsevier Science, Oxford (in press); Gershenzon et al. (1989) Plant Physiol. 89:1351-1357; Pichersky et al. (1994) Plant Physiol. 106:1533-1540. Unlike the mechanistically-related prenyltransferases farnesyl diphosphate (FPP) synthase and geranylgeranyl diphosphate (GGPP) synthase, which produce GPP as intermediates and which are nearly ubiquitous (Ogura et al. (1997) In "Dynamic Aspects of Natural Products Chemistry" (Ogura, K. and Sankawa, U., eds.), Kodansha/Harwood Academic Publishers, Tokyo, pp. 1-23), GPP synthase is largely restricted to plant species that produce abundant quantities of monoterpenes.
Because both farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase produce only negligible levels of GPP as a free intermediate on route to FPP and GGPP (Ogura et al. (1997) In "Dynamic Aspects of Natural Products Chemistry" (Ogura, K. and Sankawa, U., eds.), Kodansha/Harwood Academic Publishers, Tokyo, pp. 1-23), it is geranyl diphosphate synthase that provides the crucial link between primary metabolism and monoterpene biosynthesis and that serves as the essential driver of monoterpene biosynthesis (Wise et al. (1997) In "Comprehensive Natural Products Chemistry: Isoprenoids, Vol. 2" (Cane, D. E., ed.), Elsevier Science, Oxford (in press)).
GPP synthase has been isolated from several plant sources, including grape, geranium, sage (Croteau et al. (1989) Arch. Biochem. Biophys. 271:524-535; Heide et al. (1989) Arch. Biochem. Biophys. 273:331-338; Suga et al. (1991) Phytochemistry 30:1757-1761; Clastre et al. (1993) Plant Physiol. 102:205-211); however, the enzyme has not been characterized in any detail, and only the enzyme from grape has been purified to homogeneity.
A cDNA encoding 4S-limonene synthase from oil glands of spearmint has been described in Colby et al. (1993) J. Biol. Chem. 268(31): 23016-23024 and is available in the Genbank.TM./EMBL database and identified by the accession number L13459.
cDNAs encoding S-linalool synthase from Clarkia breweri have been described in WO 97/15584, along with methods directed at using the cDNA for enhancing the flavor and smell of plants; and in Dudareva et al. (1996) Plant Cell 8 (7): 1137-1148, also available in the Genbank.TM./EMBL database and identified by the accession number 1491939.
To exploit recombinant methods to increase monoterpene yield in monoterpene-producing species, or to genetically engineer the monoterpene biosynthetic pathway into non-producing species, it would be highly beneficial to manipulate a GPP synthase gene. Accordingly, the invention relates to expressing GPP synthase in combination with selected monoterpene synthases such as (-)-limonene synthase, S-linalool synthase, and subsequent and related pathway enzymes for production of the corresponding monoterpene product(s).